The elucidation of molecular alterations that occur during human breast cancer development may permit the identification of preventative strategies for women at high risk. Lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) confer 4- and 8-10 fold increases in risk for the development of invasive carcinoma, respectively. Our goal is to identify proteins whose expression levels vary between normal ductal/lobular units and DCIS lesions using laser capture microdissection (LCM) and proteomics.To date, six specimens have been analyzed, resulting in >300 spots that have been sent out for mass spectroscopy sequencing. Briefly, sections of flash frozen surgical specimens are subjected to LCM to purify epithelial cells from normal ducts and from DCIS. Approximately 30,000 LCM "shots" are run on two-dimensional gels, and spots of varying intensities between the two samples are excised for sequencing. To date, over 100 sequences are available. They include a number of proteins previously reported to be up-regulated in DCIS (hsp27). Many proteins identified, however, have not been linked to DCIS or breast cancer (S100A11). Signal transduction proteins have been identified (transgelin, phosphoethanolamine binding protein, tropomyosin, prohibitin) as well as several proteins involved in genomic stability/transcriptional regulation (hnRNP-L and X, high mobility group protein 1, helix destabilizing protein). Two sequences from the same specimen suggest that the intracellular vesicular trafficking process may be altered in DCIS (rab11). A number are new sequences. From these data, selected proteins will be validated by western, lysate array and possibly immunohistochemistry on a small cohort of independent normal/DCIS specimens.